Table2_Construction of a HOXA11-AS-Interacted Network in Keloid Fibroblasts Using Integrated Bioinformatic Analysis and in Vitro Validation.XLSX

Background: Expression of the long noncoding RNA (lncRNA) HOXA11-AS significantly increased in keloids by unclarified molecular regulation mechanisms.Methods: Using successfully primary cultured keloid-derived fibroblasts from central region of chronic keloid tissues (sample 0), small interfering RNAs were designed and transfected into two keloid fibroblast samples (samples 1 and 2) to knockdown HOXA11-AS. One nonspecific transfection control (sample 3) and one blank control (sample 4) were used to remove nonspecific overlap from the studied group. The lncRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs) of five samples were sequenced to identify differentially expressed (DE) profiles in HOXA11-AS-knockdown keloid fibroblasts in samples 1 and 2 (by intersection), which facilitated removal of overlap with the nonspecific controls (samples 3 and 4, by union). Using stepwise bioinformatic analysis, a HOXA11-AS-interacted competing endogenous network (ceRNA) was screened based on three DE profiles.Results: Keloid fibroblasts with or without HOXA11-AS as well as with or without nonspecific interferences were successfully constructed respectively. A total of 1,396 mRNAs and 39 lncRNAs were significantly changed in keloid fibroblast with HOXA11-AS knockdown. Simultaneously, 1,626 mRNAs and 99 lncRNAs were significantly changed in keloid fibroblast with nonspecific interference. With removal of nonspecific overlap, a lncRNA–mRNA interactive network characterized by close natural/intronic antisense relationship was initially constructed in keloid fibroblast with HOXA11-AS knockdown. Based on this network, a lncRNA–mRNA–protein interaction network was extended by integration of the human protein–protein interaction network. Significant functional genes were screened using PageRank algorithm in the extended network. Three genes, including SNED1, NIPAL3, and VTN, were validated by real-time PCR in HOXA11-AS-knockdown keloid fibroblasts. Only NIPAL3 was predicted to be a target gene for HOXA11-AS via three competing endogenous miRNAs (hsa-miRNA-19a-3p, hsa-miR-141-3p, and hsa-miR-140-5p).Conclusion: An interactive network of HOXA11-AS–three miRNAs–NIPAL3 was predicted in keloid fibroblasts by integrative bioinformatic analysis and in vitro validation.

背景：长非编码RNA（lncRNA）HOXA11-AS在瘢痕疙瘩中表达显著增加，其分子调控机制尚不明确。方法：利用从慢性瘢痕疙瘩组织中心区域成功原代培养的瘢痕疙瘩来源成纤维细胞（样本0），设计并转染小干扰RNA至两个瘢痕疙瘩成纤维细胞样本（样本1和2）以敲低HOXA11-AS。设置一个非特异性转染对照组（样本3）和一个空白对照组（样本4），以排除研究组中的非特异性干扰。对五个样本的lncRNA、信使RNA（mRNA）和microRNA（miRNA）进行测序，以识别样本1和2中HOXA11-AS敲低瘢痕疙瘩成纤维细胞的差异表达（DE）谱（通过交集），从而排除与非特异性对照组（样本3和4，通过并集）的重叠。通过逐步生物信息学分析，基于三个DE谱筛选出HOXA11-AS互作竞争性内源网络（ceRNA）。结果：分别成功构建了具有或不具有HOXA11-AS以及具有或不具有非特异性干扰的瘢痕疙瘩成纤维细胞。在HOXA11-AS敲低的瘢痕疙瘩成纤维细胞中，共1,396个mRNA和39个lncRNA表达显著改变。同时，在具有非特异性干扰的瘢痕疙瘩成纤维细胞中，共1,626个mRNA和99个lncRNA表达显著改变。通过排除非特异性重叠，在HOXA11-AS敲低瘢痕疙瘩成纤维细胞中，首先构建了一个以紧密的天然/内含子反义关系为特征的lncRNA-mRNA互作网络。基于此网络，通过整合人类蛋白质-蛋白质互作网络，扩展了lncRNA-mRNA-蛋白质互作网络。在扩展网络中，使用PageRank算法筛选出显著功能基因。在HOXA11-AS敲低瘢痕疙瘩成纤维细胞中，通过实时PCR验证了包括SNED1、NIPAL3和VTN在内的三个基因。其中，NIPAL3被预测为HOXA11-AS的三种竞争性内源miRNA（hsa-miRNA-19a-3p、hsa-miR-141-3p和hsa-miR-140-5p）的目标基因。结论：通过整合生物信息学分析和体外验证，预测了瘢痕疙瘩成纤维细胞中HOXA11-AS-三种miRNA-NIPAL3的互作网络。